The major thrust of this proposal is a genetic and biochemical analysis of the regulation of plasmid DNA replication and the extrachromosomal state of plasmid elements in bacteria with particular emphasis on the nature and mechanism of action of plasmid specified proteins and plasmid nucleotide sequences involved in the regulated initiation of plasmid DNA replication and the segregation of plasmid elements during cell division. A related problem, the genetic and biochemical mechanisms of plasmic incompatibility (i.e., the failure of certain plasmid elements to co-exist stably in a bacterial cell) also will be stressed. Efforts will be concentrated on plasmids R6K (multi-copy antibiotic resistance plasmid of narrow host range) and RD2 (limited copy number per cell and broad host range). Studies will be carried out in vitro and in vivo with Escherichia coli cells and E. coli extracts. In the case of RK2 the in vivo plasmid replication and maintenance studies will be extended to bacteria distantly related to E. coli. An analysis of the incompatibility regions of plasmid mini-F also will be continued. Finally, plasmid Co1E1 and its derivatives will be used extensively in the construction by recombinant DNA techniques of joint replicons with R6K, RK2 and mini-F and their derivatives in the analysis of regulatory mechanisms for initiation of replication, segregation of plasmids and plasmid incompatibility. Extensive analyses of regions essential for replication and incompatibility properties will be carried out at the levels of plasmid DNA reconstructions using recombinant DNA techniques, isolation and characterization of plasmid replication mutants, nucleotide sequence analysis, characterization of plasmid specified proteins involved in the regulation of plasmid replication and incompatability. A broad host range, plasmid cloning vehicle derived from plasmid RD2 will be further developed using recombinant DNA procedures. Finally, efforts will be made to develop autonomous circular DNA replicons in animal cells grown in tissue culture.